CN114990243B - 一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合和筛选半滑舌鳎抗病个体的方法 - Google Patents
一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合和筛选半滑舌鳎抗病个体的方法 Download PDFInfo
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Abstract
本发明提供了一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合和筛选半滑舌鳎抗病个体的方法,属于水产生物技术领域。所述标记组合包括微生物标记和宿主基因标记;所述微生物标记包括Alicyclobacillus pohliae、Phaeobacter inhibens和Propionibacterium acnes;所述宿主基因标记包括soat、meso1等基因。采用本发明提供的标记组合和筛选方法能够区分抗病和不抗病的半滑舌鳎个体,进而能够筛选抗病力强的半滑舌鳎优质良种。
Description
技术领域
本发明属于水产生物技术领域,尤其涉及一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合和筛选半滑舌鳎抗病个体的方法。
背景技术
半滑舌鳎(Cynoglossus semilaevis)隶属于鲽形目、舌鳎科、舌鳎属,是我国重要的海水经济养殖鱼类,其味道鲜美,营养价值高,深受广大消费者喜爱。然而,近年来,随着半滑舌鳎养殖业的迅速发展,缺乏优良品种、种质退化等问题突出;同时,养殖规模的扩大、养殖模式集约化程度的提高导致养殖病害频繁发生,尤其是以哈维氏弧菌为代表的细菌性病原引发的感染性弧菌病,导致半滑舌鳎苗种死亡率高达50%~90%,严重阻碍了半滑舌鳎养殖产业的绿色可持续发展。抗生物等药物在一定程度上可以防治病害,但长期使用可能引起耐药性并造成环境污染,进而影响人类健康。因此,开展半滑舌鳎抗病免疫的分子机制研究,筛选抗病优良种质,进而培育抗病高产新品种,对于半滑舌鳎养殖产业的发展至关重要。
近年来,在半滑舌鳎中陆续开展了抗病功能基因鉴定及表达模式分析、抗哈维氏弧菌病性状GWAS分析、转录组比较等研究,鉴定了一系列抗病相关SNP位点、差异表达基因和免疫相关的代谢通路,研究结果显示半滑舌鳎抗哈维氏弧病性状是由微效多基因调控的复杂性状,免疫通路基因、信号转导分子、代谢相关基因、细胞因子等均参与调控半滑舌鳎的抗病和免疫反应。研究显示,肠道菌群与鱼类的疾病发展、免疫系统响应之间具有密切联系,同时,肠道菌群的结构和组成也是重要的疾病诊断指标。因此,开展半滑舌鳎抗病相关基因表达调控、肠道菌群差异和宿主-菌群相互作用研究,寻找并应用能够准确区分半滑舌鳎抗病和不抗病个体的肠道微生物和宿主基因生物标记,在半滑舌鳎抗病种质筛选和良种培育中具有重要的研究意义和应用价值。
发明内容
有鉴于此,本发明的目的在于提供一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合和筛选半滑舌鳎抗病个体的方法,采用本发明提供的标记组合和筛选方法能够区分抗病和不抗病的个体,进而能够筛选抗病力强的半滑舌鳎优良种质。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合,所述标记组合包括微生物标记和宿主基因标记;
所述微生物标记包括Alicyclobacillus pohliae、Phaeobacter inhibens和Propionibacterium acnes;;
所述宿主基因标记包括soat、meso1、hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和ak基因。
本发明还提供了上述技术方案所述的标记组合筛选半滑舌鳎抗弧菌病个体的方法,包括以下步骤:
1)使用宏基因组测序方法获得候选半滑舌鳎个体肠道Alicyclobacilluspohliae、Phaeobacterinhibens和Propionibacterium acnes三种微生物的丰度信息;
2)使用转录组测序方法获得候选半滑舌鳎肠道组织soat、meso1、hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和akt基因的表达量信息;
3)使用步骤1)得到的三种微生物的丰度信息和步骤2)得到的宿主基因的表达量信息构建微生物和宿主基因组合的随机森林预测模型,区分候选半滑舌鳎是抗病个体还是不抗病个体。
本发明的有益效果:
采用本发明提供的标记组合和筛选方法能够区分抗病和不抗病的半滑舌鳎个体,进而能够筛选抗病力强的半滑舌鳎优质良种,可加快半滑舌鳎抗病良种培育,可促进鱼类抗病育种和养殖产业高质量发展,具有重要的应用价值和广阔的应用前景。使用标记组合筛选半滑舌鳎抗弧菌病个体特异性高、敏感性强,具有良好效果,可以作为潜在的标志物用于筛选半滑舌鳎抗弧菌病个体。
附图说明
图1为半滑舌鳎抗病和不抗病个体的微生物群落组成及相对丰度;
图2为半滑舌鳎抗病和不抗病个体肠道转录组比较分析;
图3为筛选抗哈维氏弧菌病半滑舌鳎的肠道微生物和宿主基因标记组合的受试者工作特征曲线(ROC)。
具体实施方式
本发明提供了一种筛选抗哈维氏弧菌病半滑舌鳎的标记组合,所述标记组合包括微生物标记和宿主基因标记;
所述微生物标记包括Alicyclobacillus pohliae、Phaeobacter inhibens和Propionibacterium acnes;;
所述宿主基因标记包括soat、meso1、hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和akt基因。
在本发明中,所述soat基因为甾醇O-酰基转移酶基因,所述meso1基因为甲基固醇单加氧酶1基因,所述hsd17b3基因为17β-羟类固醇脱氢酶3基因,所述cyp27a1基因为胆固烷三醇27-单加氧酶1基因,所述acot1_2_4基因为酰基辅酶A硫酯酶1/2/4基因,所述cyp2b基因为细胞色素P450酶2B基因,所述cyp19a基因为细胞色素P450酶19A基因,所述irf3基因为干扰素调节因子3基因,所述rpc11基因为RNA聚合酶ⅢC11亚单位基因,所述elovl5基因为超长链脂肪酸延伸蛋白5基因,所述akt基因为丝氨酸/苏氨酸蛋白激酶基因。
本发明还提供了上述技术方案所述的标记组合筛选抗哈维氏弧菌病半滑舌鳎个体的方法,包括以下步骤:
1)使用宏基因组测序方法获得候选半滑舌鳎个体肠道Alicyclobacilluspohliae、Phaeobacterinhibens和Propionibacterium acnes三种微生物的丰度信息;
2)使用转录组测序方法获得候选半滑舌鳎肠道组织soat、meso1、hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和akt基因的表达量信息;
3)使用步骤1)得到的三种微生物的丰度信息和步骤2)得到的宿主基因的表达量信息构建微生物和宿主基因组合的随机森林预测模型,区分候选半滑舌鳎是抗病个体还是不抗病个体。
本发明对使用宏基因组测序方法获得候选半滑舌鳎个体肠道Alicyclobacilluspohliae、Phaeobacterinhibens和Propionibacterium acnes三种微生物的丰度信息的方法没有特殊限定,本领域技术人员采用常规方法即可。
本发明对使用转录组测序方法获得候选半滑舌鳎肠道组织soat、meso1、hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和akt基因的表达量信息的方法没有特殊限定,本领域技术人员采用常规方法即可。
本发明对使用三种微生物的丰度信息和宿主基因的表达量信息构建微生物和宿主基因组合的随机森林预测模型的构建方法没有特殊限定,本领域技术人员采用常规的构建随机森林预测模型的方法即可,根据随机森林预测模型的结果可直接判断候选滑舌鳎是抗病个体还是不抗病个体。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
筛选与半滑舌鳎抗哈维氏弧菌病相关的肠道微生物:
1.半滑舌鳎家系构建和培养
采用陈松林等以前发明的方法建立半滑舌鳎家系(CN201010221790.8半滑舌鳎家系构建及优良家系选育方法)。
2.哈维氏弧菌人工感染半滑舌鳎实验
当半滑舌鳎幼体的全长达到8-12厘米时,通过腹腔注射方法,每个家系随机选取80-100尾鱼苗,使用哈维氏弧菌以半致死浓度(5×105CFU/g)进行人工感染。感染后每天记录鱼苗的死亡/存活个体数量,在所有家系停止死亡后(感染后14-20天)统计各家系的累计存活率,将存活率>80%和<30%的家系分别鉴定为抗病家系和不抗病家系,抗病和不抗病家系中个体分别为抗病个体和不抗病个体。
3.肠道微生物宏基因组测序
分别收集11个抗病个体和9个不抗病个体的肠道内容物样本,使用QIAamp DNAStool Mini Kit(Qiagen,Germany)DNA提取试剂盒,根据说明书提供的标准方法提取肠道内容物样本的总DNA。质量检查后,使用NEB NextUltrIlUNA文库制备试剂盒(NEB,USA),根据说明书提供的标准方法构建双端测序文库。在Illumina HiSeq XTen测序平台上,完成了17个样本的DNA测序,产生了128.04Gb的原始测序数据。使用FastQC(v0.11.6)进行原始测序数据质量评估,然后使用Trimmomatic(v0.38)软件进行序列过滤,去除读长小于100碱基的序列、接头序列、两端碱基质量小于Phred质量值20的序列和每五个碱基平均质量小于25的序列。质量控制后,获得约121.90Gb高质量宏基因组测序数据。
4.肠道菌群物种差异分析
采用MetaPhlAn2(v2.6.0)默认参数对高质量的reads进行注释,获得肠道微生物的种类组成和相对丰度信息。使用R(v4.0.3)diversity()函数计算基于香农指数的alpha多样性。采用Wilcoxon检验检测半滑舌鳎抗病和不抗病个体间肠道微生物群的差异。
5.结果
通过肠道微生物宏基因组学分析,发现抗病和不抗病个体的肠道微生物种类组成和物种丰度存在差异,其中Propionibacterium acnes(物种丰度2.85%vs.1.77%)和Phaeobacter inhibens(0.08%vs.0.01%)在抗病个体中丰度更高;而Alicyclobacilluspohliae(4.11%vs.3.61%)在易感个体中丰度更高(图1)。
实施例2
鉴定半滑舌鳎抗病和易感家系肠道差异表达基因:
1.RNA提取
分别收集11个抗病个体和9个不抗病个体的肠道组织样品。使用Trizol从肠道组织中提取总RNA。具体步骤为:取黄豆粒大小组织,经液氮研磨,放入液氮预冷的1.5ml无酶离心管,加入1ml Trizol震荡至组织充分溶解在Trizol中,4℃静置2-10min,4℃12000rpm/min离心15min。上清液加200μl(1/5Trizol体积)三氯甲烷(氯仿,预冷,避光),大力颠倒40下约15s,4℃静止5min,4℃12000rpm/min离心10min。上清液置新1.5ml离心管,加250μl异丙醇(与样品等体积,预冷),轻微颠倒40下,静止2min,4℃,12000rpm/min离心10min。弃上清,加入75%冰乙醇(现配现用)1ml,摇动,4℃静置5min,12000rpm/min离心2-5min。吸去乙醇,在室温下放置1-4min,使RNA干燥。加入DEPC水20-50μl溶解,测定浓度进行电泳检测。
2.肠道转录组测序和差异表达基因鉴定
利用提取的总RNA,使用Truseq mRNARNA-Seq文库制备试剂盒(Illumina,USA)构建双端RNA-Seq文库,文库双端序列长度为2×150bp、插入片段大小为380bp。在IlluminaHiSeq XTen测序平台上,测序文库进行测序。用RNA-QC-Chain软件对原始测序短序列的质量进行评估和过滤,去除接头序列、污染序列和低质量的短序列。使用HISAT2(v2.1.0)将过滤后的短序列与半滑舌鳎参考基因组(NCBI登记号GCA_000523025.1)进行序列比对。利用StringTie(v1.3.6)估计基因的表达水平(用FPKM表示)。
使用DESeq2(v.3.4)软件检测抗病和易感个体间的差异表达基因,其定义为|log2(FoldChange)|>1,校正后的p<0.05的基因。采用基于Benjamini-hochberg(BH)方法的错误发现率(FDR)多重检验来调整p值。使用KOBAS(v3.0.3)开展差异表达基因的KEGG代谢通路富集分析。
3.结果
通过抗病和不抗病个体宿主肠道组织转录组比较分析,鉴定到245个差异表达基因,包括抗病个体中133个上调表达基因和112个下调表达基因(图2)。富集分析显示上调表达的基因显著富集在脂质代谢通路,如甾醇和甾醇激素合成、胆固醇代谢和不饱和脂肪酸生物合成等(p<0.05),其中包含soat、meso1、cyp27a1、hsd17b3、acot1_2_4、cyp2b、cyp19a、elovl5等基因。而下调表达基因显著富集在免疫相关信号通路(p<0.05),包括Toll样受体信号通路、RIG样受体信号通路和胞质DNA-sensing通路等,其中包含irf3、rpc11、akt等基因。
实施例3
构建区分抗病和易感家系的随机森林预测模型
1.为区分抗病和易感家系,使用R包“randomForest”开发随机森林模型。为了避免过拟合,使用70%的样本进行训练,30%的样本进行测试。根据随机森林模型的均值下降精度(MeanDecreaseAccuracy)和均值下降基尼系数(MeanDecreaseGini),选择在个体预测中表现突出的微生物和宿主基因标记作为标记组合。具体为:
第一,使用含Alicyclobacillus pohliae、Phaeobacter inhibens和Propionibacterium acnes三种微生物和soat、meso1、hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和akt等11个宿主基因的标记组合,构建微生物和宿主基因组合的随机森林预测模型,以微生物的物种丰度和宿主基因的表达量作为输入文件,抗性个体和易感个体作为训练标签,以样本量7:3划分训练集和测试集。然后,根据模型的最小袋外错误率来确定mtry,再根据mtry和错误率达到稳来确定使用的随机森林的树(ntree)的数量。
第二,在mtry和ntree确定之后,对模型进行训练和预测,计算模型的准确性,使用R的pROC分析绘制受试者工作曲线(ROC),计算曲线下面积(AUC)和F1 score,来评估模型的预测能力。
2.结果:ROC曲线分析显示,AUC为0.719,准确率为0.81±0.03,F1评分为0.95,如图3所示。说明使用所述3种微生物和11个宿主基因的标记组合特异性高、敏感性强,具有良好效果,可以作为生物标记对半滑舌鳎个体抗弧菌病能力进行预测和区分。
3.在水产养殖应用中,可对待检测个体的3种微生物丰度和11个基因表达量进行定量,用所述的随机森林模型进行预测,即可获得每一条鱼是抗性个体还是易感个体,进而可以筛选出高抗个体。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (1)
1.抗哈维氏弧菌病半滑舌鳎的标记组合筛选抗哈维氏弧菌病半滑舌鳎个体的方法,所述抗哈维氏弧菌病半滑舌鳎的标记组合由肠道微生物Alicyclobacillus pohliae、Phaeobacter inhibens、Propionibacterium acnes以及肠道组织基因soat、meso1、 hsd17b3、cyp27a1、acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5、akt组成,其特征在于,包括以下步骤:
1)使用宏基因组测序方法获得候选半滑舌鳎个体肠道Alicyclobacillus pohliae、Phaeobacter inhibens和Propionibacterium acnes三种微生物的丰度信息;
2)使用转录组测序方法获得候选半滑舌鳎肠道组织soat、meso1、hsd17b3、cyp27a1、 acot1_2_4、cyp2b、cyp19a、irf3、rpc11、elovl5和akt基因的表达量信息;
3)使用步骤1)得到的三种微生物的丰度信息和步骤2)得到的宿主基因的表达量信息构建微生物和宿主基因组合的随机森林预测模型,区分候选半滑舌鳎是抗病个体还是不抗病个体。
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